Replication of positive-strand RNA viruses requires processing of virus-encoded polyproteins by viral proteases. Proteases mediate temporal and hierarchical processing of constantly changing polyprotein precursors, which in turn determine the successful viral replication and pathogenesis. Proteases of RNA viruses therefore represent important targets for development of broadly applicable inhibitors of virus replication. Rational design of protease inhibitors has targeted conserved structure/function elements such as active-site cavities and substrate-binding domains. However, RNA viruses have shown the capacity to select for resistance to these types of inhibitors. Thus, it is important to identify determinants of protease activity that are independent from catalytic or substrate sites, and which can be targeted by inhibitors that can prevent emergence of resistance. The goals of this proposal are to use the coronavirus murine hepatitis nsp5 protease as a model to: 1) test the role of nsp5 precursors, and non-catalytic, non-substrate binding structure-function determinants, in protease activity and specificity; and 2) identify and test the role of novel intramolecular residue networks in nsp5 polyprotein processing in vitro and during virus replication in culture. Coronaviruses are positive-strand RNA viruses, important pathogens of humans, and express a polyprotein composed of sixteen nonstructural replicase protein domains (nsp1-16), of which nsp4-16 are processed by the nsp5 protease (3CLpro, Mpro) at eleven cleavage sites. Nsp5 possesses two chymotrypsin-like domains, whose interface comprises the active-site cavity and substrate-binding regions, and a unique domain 3 of unknown function. In vitro biochemical and structural studies have concluded that functional nsp5 dimeric, and have identified structure/function determinants of dimerization and catalysis, but none of these have been tested in a replicating virus. Specific Aims 1 and 2 will test predicted nsp5 structure/function determinants in the context of intermediate precursors and membrane-associated complexes. Experiments in Specific Aim 3 will perform bioinformatic analysis of extensive sequence and structure datasets of nsp5 and related proteases, in order to predict intramolecular residue networks based on: a) iterative mutation and reversion of conditional temperature sensitive mutant viruses; b) co-evolution and structural proximity; and c) protein flexibility and movement. The role of predicted network residues will be tested in reverse genetic replication studies of mutant viruses, and by in vitro biochemical assays of purified nsp5. The proposed studies will answer fundamental questions in coronavirus polyprotein processing and will define critical new intramolecular networks, communication pathways, and determinants of the evolution and function of nidovirus nsp5 and orthologs. The outcome of the proposed experiments will be of high impact and significance by establishing universally applicable systems for identification and testing of novel non-catalytic RNA virus protease targets for inhibition or attenuation of virus replication.